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• W1772956064 abstract "Article15 July 2011Open Access Multiple modalities converge on a common gate to control K2P channel function Sviatoslav N Bagriantsev Sviatoslav N Bagriantsev Cardiovascular Research Institute, University of California, San Francisco, CA, USA Search for more papers by this author Rémi Peyronnet Rémi Peyronnet Institut de Pharmacologie Moléculaire et Cellulaire, UMR CNRS, Université de Nice Sophia Antipolis, Valbonne, France Search for more papers by this author Kimberly A Clark Kimberly A Clark Cardiovascular Research Institute, University of California, San Francisco, CA, USA Search for more papers by this author Eric Honoré Eric Honoré Institut de Pharmacologie Moléculaire et Cellulaire, UMR CNRS, Université de Nice Sophia Antipolis, Valbonne, France Search for more papers by this author Daniel L Minor Jr Corresponding Author Daniel L Minor Jr Cardiovascular Research Institute, University of California, San Francisco, CA, USA Departments of Biochemistry and Biophysics, and Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA, USA Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Sviatoslav N Bagriantsev Sviatoslav N Bagriantsev Cardiovascular Research Institute, University of California, San Francisco, CA, USA Search for more papers by this author Rémi Peyronnet Rémi Peyronnet Institut de Pharmacologie Moléculaire et Cellulaire, UMR CNRS, Université de Nice Sophia Antipolis, Valbonne, France Search for more papers by this author Kimberly A Clark Kimberly A Clark Cardiovascular Research Institute, University of California, San Francisco, CA, USA Search for more papers by this author Eric Honoré Eric Honoré Institut de Pharmacologie Moléculaire et Cellulaire, UMR CNRS, Université de Nice Sophia Antipolis, Valbonne, France Search for more papers by this author Daniel L Minor Jr Corresponding Author Daniel L Minor Jr Cardiovascular Research Institute, University of California, San Francisco, CA, USA Departments of Biochemistry and Biophysics, and Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA, USA Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA Search for more papers by this author Author Information Sviatoslav N Bagriantsev1, Rémi Peyronnet2, Kimberly A Clark1, Eric Honoré2 and Daniel L Minor 1,3,4,5 1Cardiovascular Research Institute, University of California, San Francisco, CA, USA 2Institut de Pharmacologie Moléculaire et Cellulaire, UMR CNRS, Université de Nice Sophia Antipolis, Valbonne, France 3Departments of Biochemistry and Biophysics, and Cellular and Molecular Pharmacology, University of California, San Francisco, CA, USA 4California Institute for Quantitative Biomedical Research, University of California, San Francisco, CA, USA 5Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA *Corresponding author. Cardiovascular Research Institute, Departments of Biochemistry and Biophysics, and Cellular and Molecular Pharmacology, University of California, San Francisco, CA 94158-9001, USA. Tel.: +1 415 514 2551; Fax: +1 415 514 2550; E-mail: [email protected] The EMBO Journal (2011)30:3594-3606https://doi.org/10.1038/emboj.2011.230 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions Figures & Info Members of the K2P potassium channel family regulate neuronal excitability and are implicated in pain, anaesthetic responses, thermosensation, neuroprotection, and mood. Unlike other potassium channels, K2Ps are gated by remarkably diverse stimuli that include chemical, thermal, and mechanical modalities. It has remained unclear whether the various gating inputs act through separate or common channel elements. Here, we show that protons, heat, and pressure affect activity of the prototypical, polymodal K2P, K2P2.1 (KCNK2/TREK-1), at a common molecular gate that comprises elements of the pore-forming segments and the N-terminal end of the M4 transmembrane segment. We further demonstrate that the M4 gating element is conserved among K2Ps and is employed regardless of whether the gating stimuli are inhibitory or activating. Our results define a unique gating mechanism shared by K2P family members and suggest that their diverse sensory properties are achieved by coupling different molecular sensors to a conserved core gating apparatus. Introduction K2P (KCNK) potassium channels produce ‘background’ currents that stabilize the membrane resting potential and that have a critical role in control of cell excitability (Bayliss and Barrett, 2008; Enyedi and Czirjak, 2010). K2Ps are members of the superfamily of voltage-gated ion channels (Yu et al, 2005) but have a unique topology; the channels are dimers of subunits that each has four transmembrane segments and two pore regions per polypeptide (Goldstein et al, 2005). Although well known as ‘leak’ channels that are constitutively open at rest, many K2Ps are polymodal and respond to a wide range of diverse regulatory inputs that include extracellular and intracellular pH, temperature, membrane stretch, polyunsaturated fatty acids, volatile anaesthetics, and noxious chemicals (Honore, 2007; Enyedi and Czirjak, 2010). Numerous studies have established that K2ps have major roles in the brain, cardiovascular system, and somatosensory neurons (Duprat et al, 2007; Honore, 2007; Bayliss and Barrett, 2008; Folgering et al, 2008; Sabbadini and Yost, 2009; Enyedi and Czirjak, 2010). Further, mutations in K2Ps have been linked to mental retardation (Barel et al, 2008) and migraine (Lafreniere et al, 2010). Nevertheless, our understanding of K2p gating mechanisms lags far behind other potassium channel classes (Cohen et al, 2009; Mathie et al, 2010) and presents a barrier to explaining how such diverse types of inputs modulate K2P function. Thus, defining K2p functional mechanisms remain an important objective for establishing how these channels influence excitation. K2P2.1 (KCNK2/TREK-1) (Fink et al, 1996; Honore, 2007) is the best-studied polymodal K2P and responds to both extracellular (Cohen et al, 2008; Sandoz et al, 2009) and intracellular (Maingret et al, 1999b; Honore et al, 2002) acidosis, heat (Maingret et al, 2000; Noel et al, 2009), mechanical forces (Maingret et al, 1999a; Noel et al, 2009), and anaesthetics (Patel et al, 1999; Heurteaux et al, 2004). K2P2.1 (TREK-1) is important in sensory neuron pain responses (Alloui et al, 2006; Noel et al, 2009) and vasodilation (Bryan et al, 2006, 2007; Blondeau et al, 2007; Garry et al, 2007). Its activity is linked to chronic pain, thermosensation (Alloui et al, 2006; Noel et al, 2009), response to general anaesthetics (Heurteaux et al, 2004), and depression (Gordon and Hen, 2006; Heurteaux et al, 2006; Perlis et al, 2008; Dillon et al, 2010). Consequently, K2P2.1 (TREK-1) along with other K2Ps present attractive targets for the development of new agents directed at treating ischaemic injury, pain, and depression (Honore, 2007; Bayliss and Barrett, 2008). In general, potassium channels contain two major points of control or ‘gates’ that are used to varied degrees to control activity depending on the particular channel (Yellen, 2002). One gate, called the ‘outer’ or ‘C-type’ gate involves the selectivity filter, which makes the direct contacts with the permeant ions, and is sensitive to external potassium concentration. The other gate, a constriction of the pore-lining transmembrane segments, is known as the ‘inner gate’ and can block access to the channel pore from the cytoplasmic side. Both K2P2.1 (TREK-1) and the drosophila K2P, KCNK0, have been shown to have a C-type gate (Zilberberg et al, 2001; Cohen et al, 2008). Because of the extracellular placement of key proton sensing residues, this type of gate has been implicated in the response of a number of K2Ps to external pH (Kim et al, 2000; Rajan et al, 2000; Lopes et al, 2001; Morton et al, 2005; Sandoz et al, 2009). Recent studies also suggest the presence of an inner gate in mutant KCNK0 channels (Ben-Abu et al, 2009), although the extent to which such a gate operates in native K2Ps is unclear. Nevertheless, the gating mechanisms that control K2P function remain obscure and many fundamental questions remain unanswered regarding the generality of these gating elements within the K2P family and how such elements are coupled to the diverse types of gating inputs (Cohen et al, 2009; Mathie et al, 2010). Such questions are magnified in the archetypal polymodal K2P K2P2.1 (TREK-1). This channel is controlled by physically diverse signals that act on different channel elements. For example, a histidine in the first extracellular loop is the main sensor for extracellular protons (Cohen et al, 2008; Sandoz et al, 2009), whereas the C-terminal cytosolic domain contains elements that are involved in the response to temperature and mechanical inputs (Patel et al, 1998; Maingret et al, 1999b, 2000; Honore et al, 2002). It has remained unclear whether these diverse modulatory inputs and sensors control K2P2.1 (TREK-1) via separate or common gating mechanisms. Additionally, because of the high sequence divergence in the K2P family, the extent to which gating mechanisms are conserved among K2Ps has remained unresolved. Here, we examine these issues with regard to external proton, heat, and pressure-evoked K2P2.1 (TREK-1) gating and demonstrate that these three gating inputs act via a common gate that has the characteristics of a C-type gate. Further, we find that regardless of whether the gating signal activates or inhibits channel function, this mechanism is conserved among diverse K2P channels. Results A yeast potassium uptake selection identifies mammalian K2P2.1 (TREK-1) gain-of-function mutations Complementation of potassium-uptake-deficient yeast has been a fruitful approach for studying a variety inward rectifier and viral potassium channels (Minor, 2009) but has not yet been applied to other types of potassium channels. Because of their ability to conduct ‘leak’ currents, we reasoned that K2P channels such as K2P2.1 (KCNK2/TREK-1) might be functional in this system. We found that unlike the inward rectifier Kir2.1 (Minor et al, 1999; Chatelain et al, 2005), mammalian K2P2.1 (TREK-1) complemented growth of a potassium-uptake-deficient yeast strain SGY1528 (Tang et al, 1995) under the mild potassium limited conditions (1 mM KCl) but not under the most stringent complementation conditions (<1 mM KCl) (Figure 1A). To identify key K2P2.1 (TREK-1) gating elements, and following precedents set with G-protein activated inward rectifiers (Sadja et al, 2001; Yi et al, 2001), we constructed a randomly mutagenized K2P2.1 (TREK-1) library that had mutations throughout the channel sequence (Supplementary Figure S1A) and selected for gain-of-function (GOF) mutants that would rescue growth in the presence of 0.5 mM KCl. The selections yielded GOF mutations in four channel regions (Figure 1B): the extracellular portion of the first selectivity filter (I148T), the second P-loop (L267P), the N-terminal part of transmembrane segment 4 (M4) (W275S and F276L), and the C-terminal cytoplasmic domain (E306G, E309A, S333G, and S333R). We also isolated a double mutant that combined the two changes in the extracellular vestibule (I148T/L267P). The identified C-terminal domain positions have been shown previously to cause K2P2.1 (TREK-1) GOF (Maingret et al, 2000; Honore et al, 2002; Murbartian et al, 2005) and thus, validate the general selection strategy. As the GOF positions in the extracellular region and the M4 positions had not been previously implicated in K2P function, we turned our attention to these. Figure 1.Functional selection identifies GOF mutations in a mammalian K2P2.1 (TREK-1). (A) Growth of potassium-transport-deficient yeast (SGY1528) expressing the yeast potassium transporter TRK1, Kir2.1, an inactive Kir2.1 mutant (Kir2.1*), K2P2.1 (TREK-1), and two exemplar K2P2.1 (TREK-1) GOF mutants, L267P and W275S under non-selective conditions (100 mM KCl), and two different selective conditions (1 mM and 0.5 mM KCl). Rows indicated with ‘Ba2+’ show growth in the presence of the Kir2.1 and K2P2.1 (TREK-1) inhibitor 8 mM BaCl2. (B) K2P2.1 (TREK-1) subunit topology diagram. Locations of GOF mutations are indicated in yellow. Transmembrane segments M1, M2, M3, and M4 and the two P-loop domains are labelled. (C) Exemplar current–voltage traces from whole cell recordings of Xenopus oocytes injected with 0.3 ng of K2P2.1 (TREK-1) or GOF mutant mRNA. Currents were elicited in solutions containing 2 mM potassium (ND96) by a ramp protocol from −150 to +50 mV from a −80 mV holding potential. Values for the average current (in μA, mean±s.e., n=5) at 0 mV were K2P2.1 (TREK-1) (0.52±0.40), I148T (1.61±0.106), L267P (1.77±0.121), I148T/L267P (3.11±0.092), W275S (4.86±0.099), F276L (1.70±0.097). (D) Quantification of normalized current amplitudes at 0 mV from Xenopus oocytes injected with 0.3 ng of mRNA for the indicated channels. (E) Cell-attached mode single channel recordings of K2P2.1 (TREK-1), I148T/L267P, and W275S expressed in COS7 cells. O1, O2, and O3, indicate the first, second, and third open states, respectively. C indicates the closed state. (F) Open channel probabilities from single channel analyses calculated on recordings of ∼30 s duration. K2P2.1 (TREK-1) (n=7), I148T (n=7), L267P (n=5), I148T/L267P (n=9), W275S (n=9), F276L (n=8). Data represent mean±s.e. Download figure Download PowerPoint Measurement of current–voltage relationships in Xenopus oocytes injected with equivalent amounts of mRNA for each of the GOF mutants or wild-type K2P2.1 (TREK-1) revealed that all tested GOF mutants had increased activity relative to wild type (Figure 1C and D). The effects of the I148T/L267P double mutant were additive compared with the individual mutants and suggest that the selectivity filter and second P-loop sites act independently. Importantly, the GOF mutants retained potassium selectivity (Supplementary Figure S1B). Three parameters affect whole cell current (I) as expressed by the equation: I=N × PO × i, where N is the active channel number, PO is single channel open probability, and i is single channel current amplitude (Hille, 2001). Therefore, we examined the effects of the GOF mutations on each of these parameters. Single channel analysis, under conditions in which similar numbers of channels were observed (Supplementary Figure S1C), demonstrated that all of the GOF mutations increase single channel open probability (mean±s.e., n=5–9): wild type (0.04±0.01), I148T (0.10±0.03), L267P (0.12±0.07), I148T/L267P (0.17±0.037), W275S (0.17±0.08), F276L (0.16±0.07) (Figure 1E and F), and that all but F276L cause a slight increase in single channel conductance (Supplementary Figure S1D and E). Furthermore, none of the tested GOF mutants increased surface expression, as judged either by biotinylation of surface proteins in COS7 cells (Supplementary Figure S2A) or by immuno-detection of the channels on the surface of the oocytes (Supplementary Figure S2B). Thus, taken together, the data indicate that the GOF mutations affected the gating machinery of the channel. Protons, heat, and mechanical force control K2P2.1 (TREK-1) activity via a common gate The K2P2.1 (TREK-1) gating apparatus includes a C-type-like outer gate that encompasses the selectivity filter that closes in response to extracellular acidosis (Cohen et al, 2008; Sandoz et al, 2009) as well as sensory elements in the C-terminal cytoplasmic tail that respond to temperature and mechanical inputs (Maingret et al, 1999b, 2000). It has remained unclear, however, whether activating stimuli such as increased temperature (Maingret et al, 2000) and mechanical force (Chemin et al, 2005) act at the same outer gate or elsewhere. To probe how the gating changes affected K2P2.1 (TREK-1) responses to both inhibitory and activating inputs, we challenged each of the GOF mutants by three different stimuli: extracellular acidosis, temperature, and mechanical stress. Strikingly, the GOF mutants I148T, L267P, I148T/L267P, and W275S affected the responses to each of the three stimuli to some degree (Figure 2; Supplementary Figure S3 and Table S1). The largest effects were caused by I148T/L267P and W275S. I148T/L267P blunted the response to all three modalities (Figure 2B, D, and F), whereas W275S had large effects on extracellular pH (pHO) and temperature responses (Figure 2B and D). In stark contrast, the reaction of the F276L GOF mutant to pHO, temperature, and mechanical stress was not significantly different from that of wild type. Even though I148T, L267P, I148T/L267P, and W275S could not be completely inhibited by pHO under the limits of our experimental conditions, their sensitivity to external magnesium inhibition (Maingret et al, 2002) remained similar to wild type (Supplementary Figure S4). This result indicates that the GOF mutations did not create channels that were generally resistant to inhibition. The fact that neither the open probability nor the surface expression of F276L exceeds that of I148T/L267P or W275S eliminates the possibility that the blunted responses of I148T/L267P and W275S arise simply because of the increased open probability or differences in expression levels. Instead, these data support the idea that the I148T, L267P, and W275S mutants act by uncoupling the sensors for the various stimuli from the gating apparatus. Further, when taken together, our data indicate that despite the radically different physical natures of the stimuli, the actions of all three modalities converge on the parts of K2P2.1 (TREK-1) in which these GOF mutations reside, the extracellular vestibule and the outer M4 region, to control gating. Figure 2.K2P2.1 (TREK-1) GOF mutations affect response to extracellular acidosis, heat, and pressure. (A) Exemplar two-electrode voltage-clamp recordings of the response of K2P2.1 (TREK-1) and the W275S GOF mutant to external pH (pHO) changes in 2 mM [K+]O solutions. (B) Normalized pHO responses (at 0 mV) for the indicated channels. (C) Exemplar two-electrode voltage-clamp recordings of the response of K2P2.1 (TREK-1) and the W275S GOF mutant to temperature in 2 mM [K+]O, pH 7.4 solutions. (D) Normalized temperature responses (at 0 mV) for the indicated channels. (E) Exemplar mechanical force (cell-attached mode, 150 mM KCl pH 7.2 in the bath, 5 mM KCl pH 7.4 in the pipette) responses of K2P2.1 (TREK-1) and the I148T/L267P GOF mutant stimulated by negative pressure applied to the extracellular side of the plasma membrane through the patch pipette. (F) Normalized pressure responses for the indicated channels. In (A, C) currents were elicited by a ramp from −150 to +50 mV from a holding potential of −80 mV. Lines for (B, D) show fits to the equations I=Imin+(Imax–Imin)/(1+([H+]O/K1/2)H) and I=Imin+(Imax–Imin)/(1+e1/2(T−T)/S), respectively. Data in (B, D, F) show mean±s.e. (n=8–30). N⩾2 for all experiments. Download figure Download PowerPoint The effect of pHO on K2P2.1 (TREK-1) is antagonized by increases in the permeant potassium ion concentration (Cohen et al, 2008; Sandoz et al, 2009) and suggests a gating mechanism that relies on conformational changes in the selectivity filter. To probe whether the GOF mutations affected this process, we measured how an increase in the extracellular potassium concentration, [K+]O, from 2 to 90 mM affected pHO inhibition. We found that I148T, I148T/L267P, and W275S had significantly diminished responses to changes in [K+]O in comparison with wild-type K2P2.1 (TREK-1) (Figure 3A and B; Supplementary Figure S5). Notably, the two GOF mutants that had the largest effects on eliminating the responses to the various gating inputs, I148T/L267P and W275S, also had the largest impact on [K+]O sensitivity (Figure 3D). Further, the pHO response of these mutants in both high and low [K+]O conditions was very similar to that of wild-type K2P2.1 (TREK-1) under high [K+]O conditions. In contrast, the F276L GOF mutant was not different than wild type (Figure 3C and D). Thus, three properties indicate that I148T/L267P and W275S GOF mutations act by stabilizing the core gating mechanism of the channel to promote the conductive conformation of the selectivity filter: the large increase in channel activity, the resistance to changes in [K+]O, and the similarity of the pHO response to that of the wild-type channel in high external potassium. These properties are consistent with a C-type gating mechanism that involves changes in the conformation of the selectivity filter. Figure 3.Extracellular loops and extracellular proximal portion of M4 control K2P2.1 (TREK-1) gating. (A–C) Normalized response to pHO in low (2 mM, data are from Figure 2B) and high (90 mM) [K+]O (2 K and 90 K, respectively) for K2P2.1 (TREK-1) and the indicated GOF mutants. Whole cell currents were elicited in Xenopus oocytes by a ramp from −150 to +50 mV from a holding potential of −80 mV (2 K) or 0 mV (90 K). (D) Quantification of the effect of high (90 mM) external potassium on the pHO response from the curves in (A–C) I6.5norm(90 K)/I6.5norm(2 K). Lines for (A–C) show fits to the Hill equation ((I=Imin+(Imax–Imin)/(1+([H+]O/K1/2)H)). Data represent mean±s.e. (n=6–30). Statistical analysis: t-test. ***P<0.001, NS, not significant (P>0.05). N⩾2 for all experiments. Download figure Download PowerPoint C-type gating of K2P potassium channels is accompanied by an increase of the relative permeability of sodium over potassium (Yuill et al, 2007; Cohen et al, 2008). To apply a further test to the idea that the conductive conformation of the pore is stabilized by the I148T/L267P and W275S GOF mutations, we measured how pHO changes affected the ion selectivity of the mutants in comparison with wild-type K2P2.1 (TREK-1) (Figure 4). Lowering pHO from 9.0 to 6.9 in a recording solution containing 100 mM sodium caused a significant right shift in the reversal potential values measured in oocytes expressing either wild-type or F276L channels (ΔErev=31.1 and 21.3 mV, respectively; Figure 4A–D) and had no effect on the reversal potential recorded in 100 mM potassium (Figure 4E). Of note, the change in the reversal potential in 100 mM sodium at pHO 6.9 did not simply arise from a decrease in the number of active potassium channels (Supplementary Figure S6). These results are consistent with prior studies of wild-type K2P2.1 (TREK-1) (Cohen et al, 2008) and are indicative of C-type gating. In contrast, under the same recording conditions, I148T/L267P and W275S were significantly less sensitive to the pHO change (ΔErev=8.8 and 12.7 mV, respectively). Accordingly, pHO changes from 9.0 to 6.9 steadily increased Na+/K+ permeability ratio of wild-type and F276L channels, but had a minimal effect on I148T/L267P and W275S (Figure 4F). These data provide further support for the idea that I148T/L267P and W275S mutations stabilize the potassium-selective conductive conformation of the selectivity filter and act on a C-type-like gate. Figure 4.Extracellular loops and extracellular proximal portion of M4 control ion selectivity of K2P2.1 (TREK-1). (A–D) Exemplar two-electrode voltage-clamp recordings of the response of the wild-type and mutant K2P2.1 (TREK-1) channel to pHO changes in 100 mM external sodium solutions. Currents were evoked by 60 ms long pulses from −150 to −50 mV in 10 mV increments from a −80 mV holding potential. Cells were injected with different amounts of mRNA to yield comparable current amplitudes. (E) Exemplar two-electrode voltage-clamp recordings of the response of the wild-type K2P2.1(TREK-1) channel to pHO changes in 100 mM external potassium solutions. Currents were evoked by 60 ms long pulses from −50 to 60 mV in 10 mV increments from a 0-mV holding potential. (F) Quantification (mean±s.e., n=8–11) of apparent permeability ratios at different pHO using the equation pNa/pK=eFΔErev/RT, where pNa and pK are permeabilities for sodium and potassium, respectively, and ΔErev is a difference between the reversal potentials measured in 100 mM sodium and 100 mM potassium solutions. N⩾2 for all experiments. Download figure Download PowerPoint The extracellular region of M4 is a key element of the K2P2.1 (TREK-1) gating apparatus Of all the GOF mutations we isolated, W275S stood out. In contrast to the Ile148 site, which is in a region that is similar to a portion of voltage-gated potassium channels that is crucial for control of the C-type gate (Lopez-Barneo et al, 1993) and the Leu267 site, which is not conserved within the K2P family, Trp275 is largely conserved in K2Ps (Supplementary Figure S7). More strikingly, it occurs in a channel element that is rather intolerant to amino-acid changes in other potassium channel classes (Collins et al, 1997; Minor et al, 1999; Irizarry et al, 2002). These observations, together with our data demonstrating that a single change at this site blunted different gating modality responses and [K+]O sensitivity, strongly suggested that this M4 region might have a special role in K2P function. As the Trp → Ser substitution causes a dramatic sidechain volume change, we tested the tolerance of the K2P2.1 (TREK-1) Trp275 position to substitution with a set of amino acids having diverse physicochemical properties. All non-aromatic substitutions examined, W275A/T/L/N/Q/D/E, resulted in channels with increased activity as assessed by two-electrode voltage-clamp (Figure 5A). In contrast, aromatic substitutions, W275Y and W275F, resulted in channels that were similar to wild type. Accordingly, investigation of how the Trp275 substitutions affect gating by pHO and temperature revealed that all non-aromatic substitutions produced channels having reduced responses to both gating stimuli (Figure 5B and C; Supplementary Table S1). These results further support the integral role of the Trp275 position in K2P2.1 (TREK-1) gating and the key role of the extracellular proximal portion of M4. Figure 5.Tests of the impact of amino-acid changes at Trp275 on K2P2.1 (TREK-1) function. (A) Normalized whole cell current amplitude (at 0 mV) recorded in 2 mM [K+]O pH 7.4 from Xenopus oocytes injected with equivalent amounts of mRNA for the indicated K2P2.1 (TREK-1) Trp275 substitutions. Statistical analysis: t-test. ***P<0.001; NS, not significant (P>0.05). (B) Normalized pHO responses in 2 mM [K+]O for the indicated Trp275 mutants. (C) Normalized temperature responses in 2 mM [K+]O for the indicated W275 mutants. Curves show fits to the Hill equation I=Imin+(Imax–Imin)/(1+([H+]O/K1/2)H) or I=Imin+(Imax–Imin)/(1+e1/2(T−T)/S). Data represent mean±s.e. (n=4–30). N⩾2 for all experiments. Download figure Download PowerPoint The extracellular region of M4 is functionally conserved across the K2P family To test whether the Trp275 equivalent position might have a general role in controlling gating within the K2P family, we examined how aromatic → Ser mutations at the analogous M4 position in other pHO-sensitive K2P channels affected function (Figure 6A). Strikingly, equivalent changes at the Trp275 homologous position in K2P10.1 (KCNK10/TREK-2), W301S; K2P9.1 (KCNK9/TASK-3), F225S; and K2P3.1 (KCNK3/TASK-1), F225S, profoundly affected pHO gating (Figure 6B–D). This effect occurred even though these pHO-gated K2P channels use different extracellular elements as [H+]O sensors (Kim et al, 2000; Rajan et al, 2000; Lopes et al, 2001; Morton et al, 2005; Sandoz et al, 2009) and was independent of whether [H+]O is inhibitory, as for K2P9.1 (TASK-3) and K2P3.1 (TASK-1), or activating, as for K2P10.1 (TREK-2). Therefore, despite diverse [H+]O-sensor placement and the non-uniform direction of gating responses to [H+]O, our data indicate that this gating stimulus converges on a common site in M4 and strongly suggests that the gates of K2P2.1 (TREK-1), K2P9.1 (TASK-3), K2P3.1 (TASK-1), and K2P10.1 (TREK-2) work by a shared mechanism. As a further test, we examined substitution of a homologous position that is not natively an aromatic residue, Glu228, in the [H+]O-gated channel K2P5.1 (KCNK5/TASK-2). E228S resulted in non-functional channels; however, introduction of an aromatic residue, E228W, profoundly inhibited K2P5.1 (TASK-2) pHO gating (Figure 6E). In accord with our K2P2.1 results, M4 mutations that make the channel less responsive to pHO gating (K2P10.1 (TREK-2) W301S and K2P5.1 (TASK-2) E228W) increase basal activity (Supplementary Figure S8). In contrast, mutations that facilitate pHO responses (K2P3.1 (TASK-1) F225S and K2P3.1 (TASK-3) F225S) suppress macro-current amplitude at pHO 7.4. Together, our studies of the effects of mutations at the K2P2.1 (TREK-1) W275 equivalent M4 position provide further evidence for the general role of this element in K2P channel gating and establish that various K2P channels utilize a conserved gating apparatus to respond to extracellular acidosis regardless of the location of the sensor or direction of the response. Figure 6.Importance of the Trp275 position is functionally conserved among K2P channels for both pHO and temperature induced gating. (A) Amino-acid alignment of the M4 region from the indicated K2P channels. Residues conserved in three or more of" @default.
• W1772956064 created "2016-06-24" @default.
• W1772956064 creator A5003450108 @default.
• W1772956064 creator A5045437602 @default.
• W1772956064 creator A5068311626 @default.
• W1772956064 creator A5079331578 @default.
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• W1772956064 date "2011-07-15" @default.
• W1772956064 modified "2023-09-25" @default.
• W1772956064 title "Multiple modalities converge on a common gate to control K_2Pchannel function" @default.
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